1. Field of the Invention
The invention relates to a novel system and method utilizing temperature sensitive markers for identifying and selecting transgenic cells, embryos, and plants.
2. Description of the Prior Art
The DNA constructs of the invention can be used to transform any type of plant cells (see below). A genetic marker must be used for selecting transformed plant cells (“a selection marker”). Selection markers typically allow transformed cells to be recovered by negative selection (i.e., inhibiting growth of cells that do not contain the selection marker) or by screening for a product encoded by the selection marker.
The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from Tn5, which, when placed under the control of plant expression control signals, confers resistance to kanamycin. Fraley et al., 1983, Proc. Natl. Acad. Sci. USA, 80:4803. Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., 1985, Plant Mol. Biol., 5:299.
Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., 1988, Plant Physiol., 86:1216, Jones et al., 1987, Mol. Gen. Genet., 210:86, Svab et al., 1990, Plant Mol. Biol., 14:197, Hille et al., 1986, Plant Mol. Biol., 7:171. Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al., 1985, Nature, 317:741-744, Stalker et al., 1988, Science, 242:419-423, Hinchee et al., 1988, Bio/Technology, 6:915-922, Stalker et al., 1988, J. Biol. Chem., 263:6310-6314, and Gordon-Kamm et al., 1990, Plant Cell, 2:603-618. Other marker genes that have been used include conditional cytotoxic genes, i.e. thymidine kinase that converts ganciclovir (an antiherpic drug) to a toxic compound (Czako and Marton, 1994, Plant Physiol., 104:1067-1071), as well as the pehA gene from Burkholderia caryophilli that codes for a phosphonate monoester hydrolase that converts glyceryl glyphosate to the herbicide glyphosate (Dotson et al., 1996, 10:383-392).
Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactate synthase. Eichholtz et al., 1987, Somatic Cell Mol. Genet., 13:67, Shah et al., 1986, Science, 233:478, Charest et al., 1990, Plant Cell Rep., 8:643, EP 154,204.
Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, R. A., 1987, Plant Mol. Biol. Rep., 5:387., Teeri et al., 1989, EMBO J., 8:343, Koncz et al., 1987, Proc. Natl. Acad. Sci. USA, 84:131, De Block et al., 1984, EMBO J., 3:1681, green fluorescent protein (GFP) (Chalfie et al., 1994, Science, 263:802, Haseloff et al., 1995, TIG, 11:328-329 and PCT application WO 97/41228). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., 1990, Science, 247:449.